SNP (Single Nucleotide Polymorphism) molecular marker related to chicken breast muscle fatty acid composition and application thereof

Abstract

The invention relates to an SNP molecular marker related to chicken breast muscle fatty acid composition and application of the SNP molecular marker, and belongs to the technical field of molecular breeding. The nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1, and the 75th basic group from the 5'end is A or T. According to the invention, through whole genome association analysis of metabolic traits PUFA / MUFA, a mutation site on the chicken METTL4 gene is found for the first time, and the mutation site is c.332Agt; t, existence of Agt; according to analysis, the site is a harmful mutation site and possibly causes the change of the function of the METTL4 gene coding protein. Experiments prove that the SNP molecular marker can be used as a molecular marker for genetic improvement of chicken varieties, is used for auxiliary breeding and molecular breeding of chickens, is beneficial to genetic improvement of chicken populations with excellent characters, and has important significance for improving the quality of chicken.

Description

Technical Field

[0001] The present invention relates to an SNP molecular marker related to the fatty acid composition of chicken pectoralis major muscle and its application, belonging to the technical field of molecular breeding. Background Art

[0002] Chicken is a traditional edible poultry and also an important model organism in scientific research. It has been an important economic poultry in China since ancient times. At present, China's chicken production and consumption rank second in the world, and chicken has become the second largest meat consumption product and an important source of animal protein in China. Chicken plays an important role in China's meat production and consumption. Therefore, the intensive and large-scale farming methods of broilers in China have also brought the growth rate of broilers to an unprecedented level. Moreover, with the improvement of living standards, people's demand for grains with more carbohydrates will gradually decrease, while the demand for meats with more protein and fat will increase significantly; the proportion of animal protein in the total protein intake will increase, and the increase in the demand for protein and fat will drive the growth of meat consumption. However, with the continuous improvement of the growth rate and feed conversion efficiency of broilers, the quality of chicken products has become increasingly prominent, making it difficult to meet the growing demand of consumers for high-quality livestock products.

[0003] A large number of studies have shown that the differential distribution of fat in chickens will have completely different effects on the quality of chicken meat. Increasing the content of intramuscular fat (IMF) in meat products can effectively increase the juiciness, tenderness of meat and enhance the flavor of meat, thereby improving the meat quality. Moreover, the ratio of the composition traits of various types of fatty acids plays a crucial role in the evaluation of the nutritional indicators of poultry meat. Modern research has shown that unreasonable dietary structures and unhealthy lifestyles are the main reasons for the prevalence of many diseases at present. Therefore, genetically improving the fatty acid composition of chicken meat to produce poultry meat products with reasonable fatty acid composition, better quality and better flavor has gradually become the main research direction of modern poultry molecular breeding.

[0004] Existing research has shown that the deposition of intramuscular fat in livestock and poultry is regulated by factors such as genetics, environment, and feed nutrition level, among which genetic factors play a crucial role in the process of intramuscular fat deposition. The heritability of chicken intramuscular fat is 0.1 - 0.16, and relevant research has shown that genetic selection can effectively increase the content of chicken intramuscular fat. Therefore, developing molecular genetic markers for chicken intramuscular fat and carrying out molecular marker-assisted selection breeding for intramuscular fat can effectively enhance the selection intensity, improve the accuracy of genetic evaluation of intramuscular fat traits, and accelerate the improvement of intramuscular fat traits.

[0005] The Chinese patent application for invention with a publication date of October 20, 2017 and a publication number of CN107267637A discloses a molecular marker related to the width of intramuscular fat in chickens and its application. The molecular marker related to the width of intramuscular fat in chickens is defined with the first base of the ACVR2B gene as the first site, and the A / T base mutation at the 86033rd site, the G / C base mutation at the 86034th site, the A / T base mutation at the 86035th site, the C / G base mutation at the 86036th site, the A / T base mutation at the 86037th site, the A / T base mutation at the 86046th site, or the T / C base mutation at the 86156th site. It also discloses primers for identifying the above molecular marker. Through the above molecular marker and identification primers, an efficient and accurate molecular marker-assisted breeding technology can be established and applied to the genetic improvement of the intramuscular fat width trait in chickens, thereby increasing the intramuscular fat width in chickens and further improving the meat quality and flavor of chicken meat.

[0006] The Chinese patent for invention with a publication date of August 29, 2023 and a publication number of CN116656838A discloses an SNP molecular marker related to intramuscular fat and body weight in chickens and its uses. Specifically, three SNP molecular markers on the exon of the LIPE gene are disclosed. Through the correlation analysis of the relevant mutation sites with the intramuscular fat content and body weight in chickens, it is found that the three SNP molecular markers on the exon of the LIPE gene are significantly correlated with the intramuscular fat content and body weight in chickens.

[0007] However, there are currently few molecular markers for breeding chickens for muscle fatty acid composition traits. Moreover, intramuscular fat content is a quantitative trait controlled by multiple genes. More molecular markers related to intramuscular fat after chicken slaughter need to be developed and used for breeding to more accurately judge intramuscular fat based on genotypes, so as to shorten the breeding process and reduce breeding costs. Summary of the Invention

[0008] The first object of the present invention is to provide an SNP molecular marker related to the fatty acid composition of chicken pectoralis major muscle and an SNP molecular marker significantly related to intramuscular fat content.

[0009] The second object of the present invention is to provide the application of the above SNP molecular marker in the assisted breeding of chicken fat traits, providing a new molecular biology means for the existing technology to accelerate the process of chicken genetic improvement.

[0010] In order to achieve the above object, the technical solution of an SNP molecular marker related to the fatty acid composition of chicken pectoralis major muscle in the present invention is as follows:

[0011] An SNP molecular marker related to the fatty acid composition of chicken pectoralis major muscle, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, and the 75th base from the 5' end is A or T.

[0012] The beneficial effects of the above technical solution are as follows: The SNP molecular marker related to the fatty acid composition of chicken pectoralis major muscle in the present invention is a pioneering invention. Through the genome-wide association analysis of the metabolic trait PUFA / MUFA, the present invention first discovers a mutation site on exon 2 of the chicken METTL4 gene, which is c.332A>T, with an A>T mutation. Through analysis, it can be known that this site is a harmful mutation site, which may lead to changes in the function of the protein encoded by the METTL4 gene and can be used as an SNP molecular marker related to the fatty acid composition of chicken pectoralis major muscle.

[0013] In order to achieve the above object, the technical solution of the application of the SNP molecular marker in the assisted breeding of chicken fat traits in the present invention is:

[0014] An application of an SNP molecular marker in the assisted breeding of chicken fat traits, wherein the fat trait is intramuscular fat.

[0015] The beneficial effects of the above technical solution are as follows: Through analysis and verification, the newly discovered SNP molecular marker on the chicken METTL4 gene in the present invention is significantly related to the fatty acid composition of chicken pectoralis major muscle. Further experiments prove that the wild-type AA genotype can significantly increase the accumulation of lipid droplets in intramuscular fat cells, while the mutant AT or TT genotype significantly inhibits the accumulation of lipid droplets in intramuscular fat cells, and the mutant AT or TT genotype inhibits the differentiation of preadipocytes in chicken pectoralis major muscle. This shows that the SNP molecular marker discovered in the present invention can be used as a molecular marker for genetic improvement of chicken breeds, for the assisted selection and molecular breeding of chickens, which is beneficial to the genetic improvement of chicken populations with excellent traits and has important significance for improving the quality of chicken meat.

[0016] As a further improvement, the genotype of the SNP molecular marker of the chicken to be tested is detected. When the genotype of the SNP molecular marker is AA, the chicken to be tested is an individual with increased intramuscular fat accumulation; when the genotype of the SNP molecular marker is AT or TT, the chicken to be tested is an individual with decreased intramuscular fat accumulation.

[0017] Specifically, due to its low fat content, high protein and essential amino acid content, chicken is recommended as the first choice of food for fitness. However, in the actual production and breeding process, broiler chickens are prone to excessive fat deposition, and most of the fat is not physiologically required. Excessive body fat deposition will hinder the normal growth and development of broiler chickens, not only wasting feed and reducing feed utilization rate, but also affecting the slaughter and processing of broiler chickens, reducing the slaughter rate and increasing economic losses. However, the increase in intramuscular fat will increase the fat content of chicken, change the nutritional ratio of chicken, and improve the quality of chicken. Therefore, in practical applications, the test chickens can be selected according to actual needs (such as: the choice of consumers and the demand of the market, etc.). If it is necessary to improve the quality of chicken, select the test chicken individuals with the AA genotype at the SNP molecular marker; if considering fitness or pursuing a healthy diet, select the test chicken individuals with the AT or TT genotype at the SNP molecular marker. The SNP molecular marker of the present invention lays a foundation for accurately and directionally breeding chickens with ideal intramuscular fat content, and is of great significance for the development of the broiler industry.

[0018] As a further improvement, select individuals with the genotype AA. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the Manhattan plot of the PUFA / MUFA trait in Experimental Example 1 of the present invention;

[0020] Figure 2 It is the linkage disequilibrium analysis module diagram in Experimental Example 1 of the present invention;

[0021] Figure 3 It is the heterozygous mutation sequencing map of the c.332A>T site of the METTL4 gene in Experimental Example 1 of the present invention;

[0022] Figure 4 It is the expression analysis of the chicken METTL4 gene and the c.332A>T site in Experimental Example 2 of the present invention (wherein, the left side is the relative expression level map of the METTL4 gene in different tissues of Gushi chickens, and the right side is the relative expression profile of the AA, AT, and TT genotypes of the c.332A>T site in the abdominal fat tissue of 14W Gushi chickens);

[0023] Figure 5In Example 3 of the experiments of the present invention, the genotype of the site c.332A>T mutation inhibits the differentiation of intramuscular preadipocytes in chicken breast muscle (wherein, A is the transfection efficiency of the overexpression of the c.332A>T wild type and mutant detected by qRT-qPCR, B and C are the Oil Red O staining results after transfection with the overexpression vectors of the c.332A>T wild type (METTL4-W) and mutant (METTL4-M), and pcDNA3.1-3xFlag (METTL4-NC) respectively, D is the detection result of the triglyceride concentration in adipocytes after transfection with the overexpression vectors of the c.332A>T wild type and mutant, and pcDNA3.1-3xFlag respectively, and E-F are the changes in the relative mRNA expression levels of adipocyte differentiation marker genes after transfection with the overexpression vectors of the c.332A>T wild type and mutant, and pcDNA3.1-3xFlag respectively). Detailed implementation manners

[0024] In the prior art, there are few molecular markers for breeding chicken muscle fatty acid composition traits, and it is necessary to dig out molecular markers with strong representative muscle fatty acid composition traits for breeding. Metabolic phenotypes are important surrogate indicators of many biological processes and pathways, including fatty acid composition. In human and animal studies, a large number of metabolic traits have been used in various omics analysis studies. Therefore, using fatty acid metabolic phenotypes for association analysis and mining molecular markers for assisting breeding of fatty acid composition traits from the perspective of genetic variation is of great significance for the improvement of fatty acid composition traits. Based on this, by means of using fatty acid metabolic phenotypes for association analysis, the present invention provides an SNP molecular marker related to the fatty acid composition of chicken breast muscle.

[0025] The present invention will be further described below in combination with the detailed implementation manners, but the protection scope of the present invention is not limited thereto; however, these embodiments are only examples and do not constitute any limitation to the scope of the present invention. Modifications or substitutions can be made to the details and forms of the present invention without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention. The test methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from conventional biochemical reagent manufacturers unless otherwise specified.

[0026] Experimental animals:

[0027] The experimental animals used in this invention are the Gushi-Anka F2 resource population. This resource population was constructed by the Henan Poultry Germplasm Resources Innovation Engineering and Technology Research Center using the local Gushi chicken breed with slow growth rate and the introduced meat-type Anka chicken breed with fast growth rate, and includes 4 orthogonal families (Anka roosters mated with Gushi hens) and 3 reciprocal cross families (Gushi roosters mated with Anka hens). Under the same feeding and management environment, they were raised in the experimental chicken farm of Henan Agricultural University, and the slaughter experiment was completed in the Poultry Genetic Improvement Laboratory of Henan Agricultural University.

[0028] The experimental reagents used in the following experimental examples are shown in Table 1, and the main instruments are shown in Table 2.

[0029] Table 1 Reagents Used in the Experiment

[0030]

[0031]

[0032] Table 2 Instruments Used in the Experiment

[0033]

[0034] The primers for RT-PCR used in the following experimental examples are shown in Table 3.

[0035] Table 3 Primers Used for qRT-PCR Analysis

[0036]

[0037]

[0038] Example of an SNP molecular marker related to the fatty acid composition of chicken pectoral muscle:

[0039] The nucleotide sequence of the SNP molecular marker related to the fatty acid composition of chicken pectoral muscle in this example is shown as SEQ ID NO.1, and the 75th base from the 5' end is A or T.

[0040] The said SNP molecular marker corresponds to the 101,193,442nd deoxynucleotide on chromosome 2 of the chicken reference genome GRCg6a version sequence information published by NCBI.

[0041] Example of the application of an SNP molecular marker in the assisted breeding of chicken fat traits:

[0042] Application of the SNP molecular marker in this embodiment in the assisted breeding of chicken fat traits, wherein the fat trait is intramuscular fat. The genotype of the SNP molecular marker of the chicken to be tested is detected. When the genotype of the SNP molecular marker is AA, the chicken to be tested is an individual with increased intramuscular fat accumulation; when the genotype of the SNP molecular marker is AT or TT, the chicken to be tested is an individual with decreased intramuscular fat accumulation. Individuals with the AA genotype of the SNP molecular marker are selected to establish a chicken population with excellent genetic resources.

[0043] Experimental Example 1 Screening and Identification of Chicken METTL4 Gene Mutation Sites

[0044] In this experimental example, gas chromatography was used to detect the content of fatty acids in the pectoral muscles of individuals in the Gushi-Anka F2 resource population, and phenotypic values of the contents of 21 fatty acids were obtained. Then, correlation analysis was performed with the SNP sites of individuals in the Gushi-Anka F2 resource population, and an SNP molecular marker related to the fatty acid composition of chicken pectoral muscles on METTL4 was obtained. The specific operation steps are as follows:

[0045] 1. Phenotype Collection

[0046] Gas chromatography was used to detect the content of fatty acids in the pectoral muscles of individuals in the Gushi-Anka F2 resource population. Among them, monounsaturated fatty acids (MUFA) include myristoleic acid (C14:1), oleic acid (C18:1), eicosenoic acid (C20:1), and erucic acid (C22:1), and polyunsaturated fatty acids (PUFA) include palmitoleic acid (C16:2), linoleic acid (C18:2), γ-linolenic acid (C18:3), 11,14-eicosadienoic acid (C20:2), 11,14,17-eicosatrienoic acid (C20:3), arachidonic acid (C20:4), 13,16,19-eicosatrienoic acid (C22:3), docosadienoic acid (C22:4), and docosahexaenoic acid (C22:6). PUFA / MUFA is the ratio of the two traits. Before performing genome-wide association analysis, logarithmic 10 transformation was performed on the PUFA / MUFA fatty acid metabolism trait so that a certain ratio and its reverse ratio could obtain the same association analysis results.

[0047] 2. Chip Scanning and Quality Control

[0048] Genomic DNA extraction was used The Blood&Tissue Handbook kit was extracted from blood samples, and the extraction steps refer to the kit instructions of QIAGEN. PE150 sequencing was performed using Illumina's HiSeq X Ten sequencing platform. The TASSEL GBS analysis pipeline (version 5.2.31) was used to identify single nucleotide polymorphisms (SNPs), and VCFtools (version 0.1.13) was used for quality control. SNPs were retained if they met the following quality control conditions: call rate > 0.30, minor allele frequency (MAF) > 0.05 (maf 0.05), quality above 98 (minGQ≥98), sequencing depth ≥ 5, following Hardy-Weinbereg equilibrium (hwe 0.0001), and retaining genotyping samples with a maximum missing rate < 0.40 for further statistical analysis. The filtered paired reads were aligned to the chicken reference genome Gallus_gallus-6.0 (released in 2018) using Bowtie2 (version 2.3.0). Before association analysis, SNPs located on sex chromosomes (ChrZ and ChrW) were removed. Finally, 323,306 SNPs of 414 F2 chickens were used for subsequent analysis.

[0049] 3. Principal Component Analysis

[0050] Population structure is the main source of confounding effects in genetic analysis. Before GWAS analysis, PCA analysis was performed using GCTA software to evaluate population structure. Considering that the high linkage disequilibrium between adjacent SNPs may bias the results of principal component analysis, all autosomal SNPs were screened to obtain independent SNPs. Then, the first two principal components were calculated and used as covariates in the mixed model. In addition, GCTA software was used to construct a genomic relationship matrix with independent SNPs and used as a random effect in the mixed model.

[0051] 4. Phenotypic Data and Basic Statistical Analysis

[0052] The mean and standard deviation of each trait were calculated using the mean and var commands in R language, and the heritability (Heritability, h2) of each trait was estimated using the reml function in the GCTA software package.

[0053] 5. Statistical Analysis Model for Genome-Wide Association Analysis

[0054] In the GCTA program, a mixed linear model (MLM) was used to perform GWAS analysis on 30 fatty acid composition traits. The following mixed linear model (MLM) was used:

[0055] y = Wα + βx + u + e

[0056] where y is the phenotypic value of each trait; W is the covariate matrix (fixed effect) including population structure (the first two PCs), sex, and batch effects; α is the vector of corresponding coefficients including the intercept; β is the SNP effect, x is the vector of SNP genotypes; u is the random effect vector with a covariance structure, following a normal distribution, i.e., u ~ N(0, KVg), where K is the known genetic relationship matrix; e is a random error vector.

[0057] Using the Bonferroni correction method, the genome-wide significance threshold was calculated using the effective number of independent SNPs. While adopting the genome-wide significance threshold (0.05 / N, where N is the number of genome-wide independent markers), we appropriately relaxed the threshold and defined the genome-wide suggestive threshold as (1 / N) in order to eliminate false negatives caused by the overly strict Bonferroni correction and thus obtain more potential SNPs associated with traits. The number of genome-wide independent markers was calculated using PLINK's -indep-pairwise with a window size of 25 SNPs and an r2 threshold of 0.1, obtaining 26,352 independent SNPs. The genome-wide significance level was set to 1.90E -06 (0.05 / 26,352; -log10(P) > 5.72), and the genome-wide suggestive threshold was set to 3.78E-05 (1 / 26,352; -log10(P) > 4.42). Manhattan and Q-Q plots were drawn using the CMplot package (https: / / github.com / YinLiLin / R-CMplot) in R software.

[0058] 6. Experimental Results and Analysis

[0059] The results of the genome-wide association analysis of the metabolic trait PUFA / MUFA showed ( Figure 1 ), a total of 11 significant SNPs were identified on chromosome 2. Haplotype analysis showed that S2_101192564, S2_101193251, and S2_101193442 were in strong linkage disequilibrium ( Figure 2 ). After annotation, it was found that S2_101193442 (c.332A>T) in the exon was a missense mutation located on the gene METTL4, resulting in the amino acid change from glutamate (GAA) to valine (GTA). The prediction score by the Sortig IntolerantFrom Tolerant (SIFT) software was 0.03, and it could be considered that c.332A>T was a harmful mutation site, which might lead to changes in the function of the protein encoded by the METTL4 gene. The sequencing chromatogram is as shown in ( Figure 3) As shown, the mutation site c.332A>T (which is the SNP molecular marker related to the fatty acid composition of chicken pectoralis major muscle in the present invention) has three genotypes (AA, AT, and TT).

[0060] Experimental Example 2 Expression analysis of chicken METTL4 gene and c.332A>T locus

[0061] In this experimental example, the expression pattern of chicken METTL4 gene and c.332A>T locus was analyzed. The specific operation steps are as follows:

[0062] 1. Experimental process

[0063] The experimental animals in this experimental example were female Gushi chickens of Chinese local breed. Three healthy individuals were selected at 14 weeks of age. After dissection, tissues such as heart, liver, spleen, lung, kidney, leg muscle, pectoralis major muscle, abdominal fat, subcutaneous fat, and pancreatic tissue were taken, frozen in liquid nitrogen, and stored at -80°C for later use.

[0064] RNA was extracted using the chloroform and isopropanol method, and then the RNA concentration and degradation degree were detected using a UV spectrophotometer and gel electrophoresis.

[0065] Reverse transcription was performed using a reverse transcription reagent kit (HiScript III RT SuperMix for qPCR(+gDNAwiper) kit). The reaction program for the first step was: 42°C for 2 min, stored at 4°C. The reaction system is shown in Table 4. The second step was set with the program: 37°C for 15 min, 85°C for 5 s, stored at 4°C. The reaction system is shown in Table 5.

[0066] Table 4 The first step of the reverse transcription system

[0067]

[0068] Table 5 The second step of the reverse transcription system

[0069]

[0070] Real-time quantitative PCR was performed using the cDNA obtained by reverse transcription as a template. The ChamQ Universal SYBR qPCR Master Mix kit was used for qRT-PCR analysis on an Applied Biosystems TM QuantStudio TM instrument. The primer sequences used are shown in Table 3. All reactions were performed with 3 biological replicates. The relative expression levels were calculated using the 2 -△△CT method. The P value was calculated using a t-test. A P value ≤ 0.05 was considered a significant difference. The data were expressed as the mean ± standard error.

[0071] All experimental data were expressed as the mean ± SEM of three replicates. The t-test method was used to calculate the significance between the two groups using SPSS version 19.0 (IBM, Chicago, IL, USA), with *p < 0.05, **p < 0.01, ***p < 0.001. GraphPad Prism 8.0 software (San Diego, CA, USA) was used for plotting.

[0072] 2. Experimental results and analysis

[0073] In this experimental example, total RNA was obtained from abdominal fat, sebum, lung, pectoral muscle, leg muscle, duodenum, kidney, liver, and heart tissue samples collected from 14-week-old Gushi chickens, and the expression level of METTL4 was detected by real-time fluorescence quantitative PCR. The results showed that METTL4 was highly expressed in abdominal fat and sebum ( Figure 4 as shown in the upper left). To investigate the effect of c.332A>T on the expression of the METTL4 gene, the expression levels of the METTL4 gene in abdominal fat tissue samples of 14-week-old Gushi chickens with three genotypes were detected. The analysis found that the expression level of the AA genotype was significantly higher than that of the AT and TT genotypes ( Figure 4 as shown in the upper right).

[0074] Functional verification of the mutation site c.332A>T in Experimental Example 3

[0075] In this experimental example, the function of the mutation site c.332A>T was verified. The specific operation steps are as follows:

[0076] 1. Isolation of primary chicken intramuscular preadipocytes

[0077] In the experiment, 2- to 3-week-old Gushi chickens were sacrificed under sterile conditions and the pectoral muscle tissue was surgically dissected in a sterile laminar flow hood. The tissue was successively washed with sterile PBS, PBS containing 1% streptomycin / penicillin, and 75% alcohol, and then washed with sterile PBS. The muscle tissue was minced with ophthalmic scissors. The tissue fragments were placed in cell digestion solution (the digestion efficiency is best when the volume ratio of the digestion solution to the tissue fragments is about 5:1), and then digested in a 37°C water bath or a 37°C constant temperature incubator for 1-1.5 h, shaking up and down every 5 min. After digestion, an equal volume of complete medium was added to terminate digestion. The filtrate was filtered through 100-mesh and 200-mesh filters respectively, and then the filtrate was centrifuged once (1000 rpm / 10 min). After removing the supernatant, an appropriate amount of erythrocyte lysate was added and lysed at room temperature for 10 min, and then centrifuged twice (1000 rpm / 10 min). After removing the supernatant again, an appropriate amount of complete medium was added to resuspend, and centrifuged three times (1000 rpm / 5 min). Finally, the cells were resuspended with an appropriate amount of complete medium and seeded into a T25 cell culture flask. The medium was changed when the cells adhered for 2-2.5 h. The adherent cells at this time were intramuscular preadipocytes.

[0078] 2. Culturing and Subculturing of Chicken Primary Intramuscular Preadipocytes

[0079] Cell culture: After isolation, chicken primary intramuscular preadipocytes were cultured in a constant-temperature cell incubator at 37 °C for 24 - 48 h. The cell density was observed using an inverted microscope. When the cell density reached 90%, subculturing was carried out. Cells from passages 3 - 6 were seeded into 6-well plates for the next experiment.

[0080] Cell subculturing: The cell density was observed using an inverted microscope. When it reached 90% and there was no bacterial or fungal contamination, subculturing could be performed. The operation was carried out in a sterile laminar flow hood. The complete medium in the culture flask was poured out, and the cells were washed 1 - 2 times with pre-warmed sterile PBS. Then, 1 mL of trypsin containing 0.25% EDTA was added and placed in a constant-temperature incubator at 37 °C for digestion for 1 - 2 min. Subsequently, 3 mL of complete medium was added to terminate the digestion. The cell suspension was placed in a 15 mL centrifuge tube and centrifuged (1000 rpm / 10 min). The supernatant was discarded, and the cells were resuspended in complete medium and then reseeded into a new cell culture flask. The flask was shaken 5 - 8 times using the "8" - shaped shaking method. After shaking evenly, it was placed in a constant-temperature incubator at 37 °C.

[0081] 3. Cell Transfection

[0082] Transfection was performed when the cell growth density in the 6-well plates reached 60% - 70%. Before transfection, first, the medium without serum and antibiotics was pre-warmed in a 37 °C water bath for 30 min. Then, according to the Lipofectamine 2000 instruction manual, the working solution was prepared. The transfection fragment and an appropriate amount of Lipofectamine 2000 were respectively added to 300 μL of Opti-DMEM and allowed to stand for 5 min. Then, the two were mixed and allowed to stand for 30 min. After that, 200 μL of the working solution was evenly added dropwise to each technical replicate in the 6-well plates. The blank control group was added with an equal amount of NC fragment or empty vector as the treatment group. After culturing in the incubator for 4 - 6 h, the medium was replaced with complete medium for subsequent experiments. Chicken intramuscular preadipocytes were transfected with different ratios of wild-type and mutant overexpression vectors of c.332A>T and Lipofectamine 2000. Taking the 12-well plates as an example, in this experiment, the dosage of Lipofectamine 2000 was fixed at 2 μL per well, and different dosages of wild-type and mutant overexpression vectors of c.332A>T (concentration diluted to 20 μM, dosages were 2 μL, 3 μL, and 4 μL per well respectively) were selected to detect the overexpression and interference efficiency, and the most appropriate dosage was selected for subsequent experiments.

[0083] 4. Oil Red Staining

[0084] Cells were seeded in 6-well cell culture plates and cultured until 80% confluence. They were then transfected with wild-type, mutant, and pcDNA3.1-3×Flag overexpression vectors respectively. After 24 h of transfection, the medium was replaced with induction differentiation medium and the cells were cultured for another 24 h. The treated cells were rinsed 2-3 times with PBS buffer, fixed with pre-cooled 4% formaldehyde for 40 min, the fixing solution was removed, and the cells were rinsed 2-3 times with PBS. Finally, they were stained with Oil Red O working solution for 20 min. The Oil Red O working solution was removed, and the cells were rinsed 2-3 times with PBS. 200 μL of PBS was added to each well to keep the cells moist, and the cells were observed and photographed under a fluorescence inverted microscope. The PBS was removed, 500 μL of 100% isopropanol was added to each well, and the mixture was allowed to stand for 10 min. The absorbance was measured at a wavelength of 500 nm using an ELISA reader.

[0085] 5. Triglyceride detection

[0086] The wild-type, mutant, and pcDNA3.1-3×Flag overexpression vectors were transfected into 6-well plates respectively. After 24 h of transfection, the medium was replaced with induction differentiation medium, and triglyceride detection was performed 24 h later. The specific operation was carried out according to the instruction manual of the enzymatic determination kit for tissue cell triglyceride (TG) content E1013 (Pulilai).

[0087] 6. Total RNA extraction, reverse transcription, and real-time fluorescence quantitative PCR of cells

[0088] The specific process and data analysis process were as described in Experimental Example 2.

[0089] 7. Experimental results and analysis

[0090] To determine the potential effect of the mutation site c.332A>T on the differentiation of chicken pectoralis major preadipocytes, wild-type and mutant vectors of c.332A>T were successfully constructed in this experimental example. The wild-type and mutant overexpression vectors were transfected into primary intramuscular preadipocytes. The expression level of METTL4 in the former increased significantly, while that in the latter decreased significantly (P<0.01) ( Figure 5 as shown in A). To accelerate the differentiation rate of intramuscular preadipocytes in vitro, induction differentiation medium was added to the transfected cells for in vitro induction differentiation. The Oil Red O staining experiment showed that overexpression of the wild-type of the mutation site c.332A>T significantly increased the accumulation of lipid droplets in intramuscular adipocytes, while the mutant significantly inhibited the accumulation of lipid droplets in intramuscular adipocytes ( Figure 5 as shown in B-C). The TG detection results showed that overexpression of the wild-type of the mutation site c.332A>T increased the TG accumulation in intramuscular preadipocytes, while overexpression of the mutant significantly inhibited the TG content in intramuscular adipocytes ( Figure 5As shown in D. qRT-PCR analysis showed that overexpression of the wild type at the mutation site c.332A>T significantly up-regulated the expression of differentiation marker genes PPARγ, C / EBPα, CEBPA, LPL, and FASN, while overexpression of the mutant type significantly inhibited the expression of the above differentiation marker genes ( Figure 5 As shown in E-F. All of the above results indicated that the genotypes AT and TT after the mutation at site c.332A>T inhibited the differentiation of intramuscular preadipocytes in chicken pectoralis major muscle.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A SNP molecular marker related to the fatty acid composition of chicken pectoralis major muscle, characterized in that: The nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1, and the 75th base from the 5' end is A or T.

2. Use of an SNP molecular marker as described in claim 1 in the assisted breeding of chicken fat traits, characterized in that: The fat trait is intramuscular fat.

3. The application of the SNP molecular marker according to claim 2 in the assisted breeding of chicken fat traits, characterized in that: Detect the genotype of the SNP molecular marker of the chicken to be tested. When the genotype of the SNP molecular marker is AA, the chicken to be tested is an individual with increased intramuscular fat accumulation; when the genotype of the SNP molecular marker is AT or TT, the chicken to be tested is an individual with decreased intramuscular fat accumulation.

4. Use of the SNP molecular marker according to claim 3 in the assistant breeding of chicken fat traits, characterized in that: Select individuals with the genotype AA.

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